Search

Summary: Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula.We biochemically characterized five flavonoid‐O‐methyltransferases (OMTs) and a lux‐based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids.We found that chalcone‐OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4′‐dihydroxy‐2′‐methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2.We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range. [ABSTRACT FROM AUTHOR]

Here, we demonstrate the beneficial effect of surfactant-producing pseudomonads on Pantoea eucalypti 299R. We conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R (Pe299R), and Pseudomonas sp. FF1 (Pff1) or Pe299R and surfactant-production deficient Pseudomonas sp. FF1::ΔviscB (Pff1ΔviscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of Pe299R when growing in the presence of Pff1 or Pff1ΔviscB. By contrast, on swarming agar plates, Pe299R was able to co-swarm with Pff1 which led to a significant increase in Pe299R biomass compared to Pe299R growing with Pff1ΔviscB or in monoculture. Finally in planta, and using the single-cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated Pe299R subpopulations that did not occur in the presence of Pff1ΔviscB. We tested three additional surfactant-producing pseudomonads and their respective surfactant knockout mutants on PE299R on swarming agar showing similar results. This led us to propose a model for the positive effect of surfactant production during leaf colonization. Our results indicate that co-motility might be common during leaf colonization and adds yet another facet to the already manyfold roles of surfactants. [ABSTRACT FROM AUTHOR]

ABSTRACTFluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells, and organisms, as well as gene regulation. Green fluorescent protein and all its derivatives are now standard tools in cell biology, immunology, molecular biology, and microbiology laboratories around the world. A common feature of these proteins is their dioxygen (O2)-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development and in cellulo characterization of genetic circuits encoding the O2-independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7-based constructions can be used to produce fluorescing cells of Vibrio diazotrophicus, a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world.IMPORTANCEFluorescent proteins are used for decades, and have allowed major discoveries in biology in a wide variety of fields, and are used in environmental as well as clinical contexts. Green fluorescent protein (GFP) and all its derivatives share a common feature: they rely on the presence of dioxygen (O2) for protein maturation and fluorescence. This dependency precludes their use in anoxic environments. Here, we constructed a series of genetic circuits allowing production of KOFP-7, an O2-independant flavin-binding fluorescent protein. We demonstrated that Escherichia coli cells producing KOFP-7 are fluorescent, both at the population and single-cell levels. Importantly, we showed that, unlike cells producing GFP, cells producing KOFP-7 are fluorescent in anoxia. Finally, we demonstrated that Vibrio diazotrophicus NS1, a facultative anaerobe, is fluorescent in the absence of O2 when KOFP-7 is produced. Altogether, the development of new genetic circuits allowing O2-independent fluorescence will open new perspective to study anaerobic processes.

The aim of this research is to define optimal conditions to improve the stability of gold and silver nanoparticles' anti-zearalenone antibody conjugates for their utilisation in lateral flow immunochromatographic assay (LFIA). The Turkevich–Frens method was used to synthesise gold nanoparticles (AuNPs), which were between 10 and 110 nm in diameter. Silver nanoparticles (AgNPs) with a size distribution of 2.5 to 100 nm were synthesised using sodium borohydride as a reducing agent. The onset of AuNP and AgNP aggregation occurred at 150 mM and 80 mM NaCl concentrations, respectively. Stable Au and Ag nanoparticle–antibody conjugates were achieved at 1.2 mM of K2CO3 concentration, which corresponds to the pH value of ≈7. Lastly, the highest degree of conjugation between Au and Ag nanoparticles and anti-zearalenone antibodies was at 4 and 6 µg/mL of antibody concentrations. The optimisation of the conjugation conditions can contribute to better stability of nanoparticles and their antibody conjugate and can improve the reproducibility of results of bioreporter molecules in biosensing lateral flow devices. [ABSTRACT FROM AUTHOR]

Since the initial introduction of whole-cell bioreporters (WCBs) nearly 30 years ago, their high sensitivity, selectivity, and suitability for on-site detection have rendered them highly promising for environmental monitoring, medical diagnosis, food safety, biomanufacturing, and other fields. Especially in the environmental field, the technology provides a fast and efficient way to assess the bioavailability of pollutants in the environment. Despite these advantages, the technology has not been commercialized. This lack of commercialization is confusing, given the broad application prospects of WCBs. Over the years, numerous research papers have focused primarily on enhancing the sensitivity and selectivity of WCBs, with little attention paid to their wider commercial applications. So far, there is no a critical review has been published yet on this topic. Therefore, in this article we critically reviewed the research progress of WCBs over the past three decades, assessing the performance and limitations of current systems to understand the barriers to commercial deployment. By identifying these obstacles, this article provided researchers and industry stakeholders with deeper insights into the challenges hindering market entry and inspire further research toward overcoming these barriers, thereby facilitating the commercialization of WCBs as a promising technology for environmental monitoring. [Display omitted] [ABSTRACT FROM AUTHOR]

The aim of this research is to define optimal conditions to improve the stability of gold and silver nanoparticles’ anti-zearalenone antibody conjugates for their utilisation in lateral flow immunochromatographic assay (LFIA). The Turkevich–Frens method was used to synthesise gold nanoparticles (AuNPs), which were between 10 and 110 nm in diameter. Silver nanoparticles (AgNPs) with a size distribution of 2.5 to 100 nm were synthesised using sodium borohydride as a reducing agent. The onset of AuNP and AgNP aggregation occurred at 150 mM and 80 mM NaCl concentrations, respectively. Stable Au and Ag nanoparticle–antibody conjugates were achieved at 1.2 mM of K2CO3 concentration, which corresponds to the pH value of ≈7. Lastly, the highest degree of conjugation between Au and Ag nanoparticles and anti-zearalenone antibodies was at 4 and 6 µg/mL of antibody concentrations. The optimisation of the conjugation conditions can contribute to better stability of nanoparticles and their antibody conjugate and can improve the reproducibility of results of bioreporter molecules in biosensing lateral flow devices.

Abstract Background Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. Results In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. Conclusions This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.

Due to the public health concerns of endocrine-disrupting chemicals, there is an increasing demand to develop improved high-throughput detection assays for enhanced exposure control and risk assessment. A substrate-free, autobioluminescent HEK293ARE/Gal4-Lux assay was developed to screen compounds for their ability to induce androgen receptor (AR)-mediated transcriptional activation. The assay was validated against a group of 40 recommended chemicals and achieved an overall 87.5% accuracy in qualitatively classifying positive and negative AR agonists. The HEK293ARE/Gal4-Lux assay was demonstrated as a suitable tool for Tier 1 AR agonist screening. By eliminating exogenous substrate, this assay provided a significant advantage over traditional reporter assays by enabling higher-throughput screening with reduced testing costs while maintaining detection accuracy.

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering. [ABSTRACT FROM AUTHOR]

DNT (2,4-dinitrotoluene), a volatile impurity in military grade TNT-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described DNT bioreporter strains, based on a fusion between the Escherichia coli yqjF gene promoter to either a bacterial luxCDABE luminescence gene cassette or to the green fluorescent protein gene gfp. We now present the azoR gene promoter, demonstrated to be strongly induced by DNT, as an alternative sensing element for this compound. An E. coli bioreporter strain harboring a plasmid-borne fusion of the azoR promoter region to Photorhabdus luminescens luxCDABE displayed stronger bioluminescent responses to DNT than the yqjF-based bioreporter. The azoR promoter was induced not directly by DNT but by its degradation products; the most potent inducer was 2,4,5-trihydroxytoluene (THT). This reaction was positively regulated by YhaJ, a member of the LysR family of transcriptional regulators, and was significantly amplified in a yhaK mutant. We also show that DNT degradation in E. coli involves glutathione, which plays a vital role in azoR gene promoter activation.

A possible solution for the standoff detection of buried landmines is based on the use of microbial bioreporters, genetically engineered to emit a remotely detectable optical signal in response to trace amounts of explosives' signature chemicals, mostly 2,4‐dinitrotoluene (DNT). Previously developed DNT sensor strains were based on the fusion of a DNT‐inducible gene promoter to a reporting element, either a fluorescent protein gene or a bacterial bioluminescence gene cassette. In the present study, a different approach was used: the DNT‐inducible promoter activates, in Escherichia coli, the quorum‐sensing luxI and luxR genes of Aliivibrio fischeri. N‐Acyl homoserine lactone (AHL), synthesized by LuxI, combines with LuxR and activates the bioluminescence reporter genes. The resulting bioreporter displayed a dose‐dependent luminescent signal in the presence of DNT. Performance of the sensor strain was further enhanced by manipulation of the sensing element (combining the E. coli DNT‐inducible azoR and yqjF gene promoters), by replacing the luminescence gene cassette of Photorhabdus luminescens luxCDABE with A. fischeri luxCDABEG, and by introducing two mutations, eutE and ygdD, into the host strain. DNT detection sensitivity of the final bioreporter was over 340‐fold higher than the original construct. [ABSTRACT FROM AUTHOR]

Characterizing waste ecotoxicity is laborious because of both the undefined nature of environmental samples and the diversity of contaminants that can be present. With regard to these limitations, traditional approaches do not provide information about the nature of the pollution encountered. To improve such assessments, a fluorescent library of 1870 transcriptomic reporters from Escherichia coli K12 MG1655 was used to report the ecotoxic status of environmental samples. The reliability of the approach was evaluated with 6 metallic pollutants (As, Cu, Cd, Hg, Pb, Zn) used alone and in mixture in pure and complex matrices. A total of 18 synthetic samples were used to characterize the specificity of the resulting metallic contamination fingerprints. Metallic contamination impacted 4.5 to 10.2% of the whole transcriptomic fingerprint of E. coli. The analysis revealed that a subset of 175 transcriptomic reporters is sufficient to characterize metallic contamination, regardless of the nature of the sample. A statistical model distinguished patterns due to metallic contamination and provided information about the level of toxicity with 93 to 98% confidence. The use of the transcriptomic assessment was validated for 17 complex matrices with various toxicities and metal contaminants, such as activated sludge, wastewater effluent, soil, wood and river water. The presence of metals and their associated toxicity, which seems linked to their bioavailabilities, were thereby determined. This method constitutes a possible tool to screen unknown complex samples for their metallic status and identify those for which a deeper characterization must be achieved by the use of traditional biosensors and analytical methods. [ABSTRACT FROM AUTHOR]

Detection of buried landmines is a dangerous and complicated task that consumes large financial resources and poses significant risks to the personnel involved. A potential alternative to conventional detection methodologies is the use of microbial bioreporters, capable of emitting an optical signal upon exposure to explosives, thus revealing to a remote detector the location of buried explosive devices. We have previously reported the design, construction, and optimization of an Escherichia coli-based bioreporter for the detection of 2,4,6-trinitrotoluene (TNT) and its accompanying impurity 2,4-dinitrotoluene (DNT). Here we describe the further enhancement of this bioreporter by the directed evolution of YhaJ, the transcriptional activator of the yqjF gene promoter, the sensing element of the bioreporter’s molecular circuit. This process resulted in a 37-fold reduction of the detection threshold, as well as significant enhancements to signal intensity and response time, rendering this sensor strain more suitable for detecting the minute concentrations of DNT in the soil above buried landmines. The capability of this enhanced bioreporter to detect DNT buried in sand is demonstrated.

Quorum sensing (QS) helps the Xanthomonas group of phytopathogens to infect several crop plants. The vascular phytopathogen Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease on Brassicaceae leaves, where a typical v-shaped lesion spans both vascular and mesophyll regions with progressive leaf chlorosis. Recently, the role of QS has been elucidated during Xcc early infection stages. However, a detailed insight into the possible role of QS-regulated bacterial invasion in host chlorophagy during late infection stages remains elusive. In this study, using QS-responsive whole-cell bioreporters of Xcc, we present a detailed chronology of QS-facilitated Xcc colonization in the mesophyll region of cabbage (Brassica oleracea) leaves. We report that QS-enabled localization of Xcc to parenchymal chloroplasts triggers leaf chlorosis and promotion of systemic infection. Our results indicate that the QS response in the Xanthomonas group of vascular phytopathogens maximizes their population fitness across host tissues to trigger stage-specific host chlorophagy and establish a systemic infection. [ABSTRACT FROM AUTHOR]

Background: Biofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation. Results: In this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain. Conclusions: This suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation. [ABSTRACT FROM AUTHOR]

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering.

Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells, and organisms, as well as gene regulation. Green fluorescent protein and all its derivatives are now standard tools in cell biology, immunology, molecular biology, and microbiology laboratories around the world. A common feature of these proteins is their dioxygen (O 2 )-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development and in cellulo characterization of genetic circuits encoding the O 2 -independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7-based constructions can be used to produce fluorescing cells of Vibrio diazotrophicus , a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world.IMPORTANCEFluorescent proteins are used for decades, and have allowed major discoveries in biology in a wide variety of fields, and are used in environmental as well as clinical contexts. Green fluorescent protein (GFP) and all its derivatives share a common feature: they rely on the presence of dioxygen (O 2 ) for protein maturation and fluorescence. This dependency precludes their use in anoxic environments. Here, we constructed a series of genetic circuits allowing production of KOFP-7, an O 2 -independant flavin-binding fluorescent protein. We demonstrated that Escherichia coli cells producing KOFP-7 are fluorescent, both at the population and single-cell levels. Importantly, we showed that, unlike cells producing GFP, cells producing KOFP-7 are fluorescent in anoxia. Finally, we demonstrated that Vibrio diazotrophicus NS1, a facultative anaerobe, is fluorescent in the absence of O 2 when KOFP-7 is produced. Altogether, the development of new genetic circuits allowing O 2 -independent fluorescence will open new perspective to study anaerobic processes., Competing Interests: The authors declare no conflict of interest.

Using a phyllosphere model system, we demonstrated that the term 'carrying capacity', as it is commonly used in microbial ecology, needs to be understood as the sum of many 'local carrying capacities' in order to better explain and predict the course and outcome of bacterial colonization of an environment. Using a green fluorescent protein-based bioreporter system for the quantification of reproductive success (RS) in individual Erwinia herbicola cells, we were able to reconstruct the contribution of individual immigrants to bacterial population sizes on leaves. Our analysis revealed that plant foliage represents to bacteria an environment where individual fate is determined by the local carrying capacity of the site where an immigrant cell lands. With increasing inoculation densities, the RS of most immigrants declined, suggesting that local carrying capacity under the tested conditions was linked to local nutrient availability. Fitting the observed experimental data to an adapted model of phyllosphere colonization indicated that there might exist three types of sites on leaves, which differ in their frequency of occurrence and local carrying capacity. Specifically, our data were consistent with a leaf environment that is characterized by few sites where individual immigrants can produce high numbers of offspring, whereas the remainder of the leaf offered an equal number of sites with low and medium RS. Our findings contribute to a bottom-up understanding of bacterial colonization of leaf surfaces, which includes a quantifiable role of chance in the experience at the individual level and in the outcome at the population level.

The Lachish River has suffered from recurring pollution incidents for the past decade. On October 11th, 2017, another contamination in the river was sighted, as thousands of dead fish were found floating. Samples from the river were retrieved and tested through a whole cell bioluminescent bacterial bioreporter system as well as conventional analytical methods, and the results from both methods were analyzed and compared, even though initially these two collecting events were not coordinated. The information acquired from the whole cell reporter was consistent with that obtained from conventional methods. Both approaches indicated a large concentration of microorganisms as deduced from K802NR E. coli strain reaction and coliforms count. The high water conductivity measured in collected samples were closer downstream, and attributed to the diffusion of salts from the Mediterranean Sea which affected bacterial viability as seen from the decreased reaction of E. coli strains TV1061 and DPD2794. In addition, the bacterial indicators’ kinetic patterns have shown indication for the presence of a genotoxic substance from only one of the collection sites, which was tested positive for the herbicide Metazachlor, itself known to have genotoxic effects. The correlation between both approaches, along with the biosensor’s ability to assess biological influences, suggests that the whole cell bioluminescent bacterial bioreporter bioassay as an easy, simple and efficient approach for water toxicity monitoring.

Background: Copper is a pollutant compound which can cause earnest toxicity in human and some organisms. Bioreporters are frugal and non-toxic detectors for pollution compounds. Precedent designed recombinant Escherichia coli copper bioreporters with the lux gene of Vibrio fischeri or Aequorin luciferase of Jellyfish does not provide a high sensitivity. The aim of current study was to design an incipient Copper bioreporter with applying firefly luciferase and Copper resistance promoter of P. syringae pv.Tomato in Escherichia coli XL1-Blue. Methods: Recombinant pGL3 was obtained by applying the pGL3-Control vector to Escherichia coli XL1-Blue, by Polymerase Chain Reaction method and double digestion. Recombinant Escherichia coli cells were cultured with applying different concentrations of copper sulphate to study the activity of luciferase by Luminometer. Copper bioreporter specificity was resolute by different concentrations of Zinc sulfate and Ferric sulfate. Results: Recombinant Escherichia coli BL21, with copper promoter gene in pGL3 Vector showed the highest Luciferase activity in 0.1 millimolar of Copper sulfate. The highest Luciferase activity was in 0.09 millimolar and 1.0 millimolar of Zinc sulfate and Ferric sulfate respectively. Conclusion: Current study provided a categorical bioreporter for detecting copper, utilizing firefly luciferase with a high specificity (96.1%). By optimizing inhibitor factors, application of current copper bioreporter can be developed in human life.

In this study, the disrupting effects of glyphosate (GLY), aminomethylphosphonic acid (AMPA), and three glyphosate-based herbicides (GBHs) on vitellogenesis in a non-concentration-dependent manner are reported for the first time in 120 h of acute exposure of zebrafish at environmentally relevant concentrations. GBHs are commonly used worldwide in weed control management. Due to their extensive application, they frequently occur in aquatic ecosystems and may affect various organisms. The active substance GLY and its major by-product, AMPA, are the most thoroughly studied chemicals; however, the adverse effects of the complex formulas of GBHs with diverse and unknown content of co-formulants are still not sufficiently researched. This study focused on the embryotoxicity, sublethal malformations, and estrogenic potency of GLY, AMPA, and four commonly used GBHs on zebrafish embryos using a wild type and an estrogen-sensitive, transgenic zebrafish line (Tg(vtg1:mCherry)). After 120 h of exposition, AMPA did not cause acute toxicity, while the LC 50 of GLY was 160 mg/L. The GBHs were more toxic with LC 50 values ranging from 31 to 111 GLY active equivalent (a.e.) mg/L. Exposure to 0.35–2.8 mg/L GBHs led to sublethal abnormalities: typical symptoms were structural deformation of the lower jaw and anomalies in the olfactory region. Deformity rates were 10–30% in the treated groups. In vivo , fluorescently expressed vtg1 mCherry protein in embryonic liver was detected by a non-invasive microscopic method indicating estrogenic action through vitellogenin production by GLY, AMPA, and GBHs. To confirm the in vivo findings, RT-qPCR method was performed to determine the levels of the estrogenicity-related vtg1 mRNA. After 120 h of exposure to GLY, AMPA, and three GBHs at a concentration of 0.35 mg/L, the expression of vtg1 gene was significantly up-regulated. Our results highlight the risk that short-term GLY and GBH exposure can cause developmental malformations and disrupt the hormonal balance in zebrafish embryos. [Display omitted] • GLY, AMPA, and GBHs have affected the embryonic stage of zebrafish lines. • In vivo estrogenicity was observed in transgenic (Tg(vtg1:mCherry)) embryos. • RT-qPCR has revealed changes in vitellogenin gene expression at 0.35 mg/L of GLY. • Malformations and estrogenicity were induced at environmental concentrations. [ABSTRACT FROM AUTHOR]

Aim: Escherichia coli is the conventional choice as the host strain for whole‐cell bioreporter construction due to its well‐understood genetics and well‐established cloning protocols. However, for real‐world environmental biosensing applications, it is often beneficial to use a bacterial strain derived directly from the environment under study to better ensure chemical target specificity and optimal response time. The aim of this study was to develop a whole‐cell bioreporter for detection of bioavailable mercury by replacing E. coli with a wild‐type bacterial host derived from a soil environment. Materials and Results: In this study, an Enterobacter cloacae strain isolated from soil derived from a municipal and electronic waste dumping site was engineered to serve as a bioluminescent bioreporter for mercury toxicity by linking its merR‐like gene and promoter sequence to a reorganized luxABCDE gene cassette from Photorhabdus luminescens. This bioreporter, designated as E. cloacae DWH4lux, detected mercury (HgCl2) at a minimum concentration of 0·2 µg l−1 with a linear response profile being maintained between a range of 0·4–1600 µg l−1 (R2 = 0·9604) with a peak bioluminescent response occurring within 1 h after exposure. No significant synergistic or antagonistic influences were observed on the bioluminescent response by other contaminating metal elements. Enterobacter cloacae DWH4lux was also demonstrated to detect mercury effectively in artificially contaminated water sample with linear correlation (R2 = 0·9623). Conclusions: The results indicated that E. cloacae DWH4lux could detect mercury in quantities below the US Environmental Protection Agency's permitted limit values (2 µg l−1). Hence, it is concluded that E. cloacae DWH4lux has the potential to serve as an effective whole‐cell bioreporter for the environmental monitoring of mercury contamination. Significance and Impact of the Study: This study provides new insight into the recruitment of mercury‐tolerant bacterial hosts derived from environmental samples over the conventional lab‐based E. coli host for the construction of mercury bioreporters. With improved response time and selectivity, the environmentally sourced bacteria can serve as an alternative host choice to improve biosensing technology in the near future. [ABSTRACT FROM AUTHOR]

Today, two types of lamp systems dominate the UV disinfection industry: low-pressure (LP) UV lamps and medium-pressure (MP) polychromatic lamps. Both lamp types have their advantages and disadvantages in microorganism inactivation, with LP lamps being cheaper, having longer life, and working at lower temperature, hence reducing fouling, and MP lamps showing better inactivation per germicidal dose for certain microorganisms. Bacterium-based biosensors were used to compare LP and MP irradiation. These biosensors were Escherichia coli bacteria carrying the lux operon genes under the control of different stress-responding promoters, where activation of the specific promoter is manifested as bioluminescence. MP irradiation, considerably more than LP irradiation, resulted in activation of the superoxide dismutase expression, indicating the formation of superoxide radicals inside the cells. Accordingly, pre-exposure (immunization) of the bacteria to an activator that produces superoxide radicals resulted in lower inactivation and increased resistance to MP irradiation, but not to LP irradiation. This study shows that the difference in germicidal efficiency may result from the production of intracellular superoxide radicals by MP irradiation, at wavelengths other than 254 nm, as emitted by LP lamps. Image 1 • MP irradiation showed better inactivation of E. coli than LP irradiation. • MP triggered much more intracellular superoxide production than LP. • Pre-exposure to superoxide resulted in elevated resistance to MP inactivation. • Biosensors as tools to study mechanisms underlying inactivation by LP and MP lamps. [ABSTRACT FROM AUTHOR]

Biological systems with intrinsic luminescent properties serve as powerful and noninvasive bioreporters for real-time and label-free monitoring of cell physiology. This study employs the bioluminescent marine bacterium Vibrio fischeri to investigate the effects of separated microwave electric (E) and magnetic (H) fields. Using a cylindrical TM010 mode aluminum resonant cavity, designed to spatially separate E and H fields of a pulsed microwave (2.45 GHz) input, we sampled at 100-ms intervals the 490-nm emission of bioluminescence from suspensions of the V. fischeri. E-field exposure (at 4.24 and 13.4 kV/m) results in rapid and sensitive responses to 100-ms pulses. H-field excitation elicits no measurable responses, even at 100-fold higher power input levels (equivalent to 183 A/m). The observed effects on bacterial light output partially correlate with measured E-field-induced temperature increases. In conclusion, the endogenous bioluminescence of V. fischeri provides a sensitive and noninvasive method to assess the biological effects of microwave fields. [ABSTRACT FROM AUTHOR]

A synthetic bacterial luciferase-based autobioluminescent bioreporter, HEK293ERE/Gal4-Lux, was developed in a human embryonic kidney (HEK293) cell line for the surveillance of chemicals displaying endocrine disrupting activity. Unlike alternative luminescent reporters, this bioreporter generates bioluminescence autonomously without requiring an external light-activating chemical substrate or cellular destruction. The bioreporter's performance was validated against a library of 76 agonistic and antagonistic estrogenic endocrine disruptor chemicals and demonstrated reproducible half maximal effective concentration (EC50) values meeting the U.S. Environmental Protection Agency (EPA) guidelines for Tier 1 endocrine disrupting chemical screening assays. For model compounds, such as the estrogen receptor (ER) agonist 17β-estradiol, HEK293ERE/Gal4-Lux demonstrated an EC50 value (7.9 × 10−12 M) comparable to that of the current EPA-approved HeLa-9903 firefly luciferase-based estrogen receptor transcription assay (4.6 × 10−12 M). Screening against an expanded array of common ER agonists likewise produced similar relative effect potencies as compared with existing assays. The self-initiated autobioluminescent signal of the bioreporter permitted facile monitoring of the effects of endocrine disrupting chemicals, which decreased the cost and hands-on time required to perform these assays. These characteristics make the HEK293ERE/Gal4-Lux bioreporter potentially suitable as a high-throughput human cell-based assay for screening estrogenic activity. [ABSTRACT FROM AUTHOR]

• Cyanobacterial nitrate bioreporters were optimized based on various combinations. • LuxABCDE responded faster than sfGFP in cyanobacterial nitrate bioreporters. • The shuttle plasmid with RSF1010 replicon improved the performance of bioreporters. • The nitrate bioavailability can be detected by bioreporter AD-AS-X at 2 h induction. • Six bioreporters effectively detecting the bioavailable nitrate in the water sample. The water eutrophication, resulting from the discharge of industrial and agricultural wastewater, leads to ecological degradation. However, to date, how to assess and manage the risks of water pollution, especially nitrogen pollution, remains a particularly noteworthy issue. Nitrate, the most important nitrogen compound, has become a bottleneck restricting total nitrogen management. The development of bioreporters monitoring nitrate pollution contributes to the estimation of water quality, especially the availability of nutrients. In this study, we obtained 9 bioreporters from 40 cyanobacterial derivatives which were constructed based on different hosts, copy numbers, and sensing elements and evaluated the performance of bioreporters. The results showed that single-celled Synechocystis was more sensitive to nitrate than filamentous Anabaena , that the reporter gene luxABCDE responded faster than sfgfp in most bioreporters, and that relatively medium-copy plasmid improved the performance of sensing elements. Nine bioreporters performed well in bioavailable nitrate detection, of which AD-AS-X and AR-NI-X, activated by nitrate repletion, had the shortest response time (2 h) and the widest response range (20–800 μM), respectively. Moreover, SR-GLN-SG, activated by nitrate deficiency, exhibited the best linear response (R2 = 0.998). After parameter optimization, exponential growth phase bioreporters, culture temperature of 30 °C, sample volume of 200 μL were determined as optimal monitoring conditions. We found that common water contaminants (copper, cadmium, and phosphorus) had no impact on the performance of bioreporters, indicating the stability of bioreporters. Six out of 9 bioreporters, especially the SR-NB-X, were highly effective in detecting the bioavailable nitrate in wastewater sample. This study provides valuable references for developing more cyanobacterial bioreporters and their practical application in nitrate detection. [Display omitted] [ABSTRACT FROM AUTHOR]

Based on the sole information of structural genes of the 2-nitrobenzoate (2NBA) utilizing catabolic gene cluster (onbX1X2FCAR1EHJIGDBX3), 2NBA-sensing bioreporters were constructed by incorporating egfp into the onb gene cluster of Cupriavidus sp. strain ST-14. Incorporation of reporter gene in proximal to the hypothesized promoter region in conjunction with the disruption of the gene encoding inducer-metabolizing enzyme was turned out to be advantageous in reporter gene expression at low inducer concentration. The bioreporter strain was capable of expressing EGFP from the very 1st hour of induction and could detect 2NBA at (sub) nanomolar level exhibiting a strict specificity toward 2NBA, displaying no response to EGFP expression from its meta- and para-isomers as well as from a number of structurally related compounds. The present study is a successful demonstration of the development of a 2NBA-sensing bioreporter with respect to ease of construction, inducer specificity, and sensitivity, without prior knowledge of the associated inducer-responsive promoter-regulator elements. The present approach can be used as a model for the development of bioreporters for other environmental pollutants.

Abstract Vibrio campbellii BAA-1116 is renowned for its bioluminescence properties, and genetic tools are available to genetically track this strain. However, many other ecologically important V. harveyi strains exist, for which only few genetic tools are available. In this study, a rapid electroporation protocol was developed to transform replicative plasmids in various environmental V. harveyi and Pseudoalteromonas strains. Moreover, a mini- Tn 7 delivery system was modified to site-specifically integrate mini-transposons in the genome of V. harveyi ORM4. As a proof-of-principle, replicative plasmids carrying bioreporters were introduced by electroporation in V. harveyi ORM4 cells, and gene expression was followed at the single cell level. We could demonstrate that a flagellar gene is subjected to bimodal gene expression in V. harveyi ORM4, being highly expressed in 10% of the population in stationary phase. This study extends the possibilities to study environmental Vibrio strains and uncovers the occurrence of phenotypic heterogeneity in flagellar expression in Vibrio. [ABSTRACT FROM AUTHOR]

An autonomously bioluminescent Saccharomyces cerevisiae BLYAhS bioreporter was developed in this study for the simple and rapid detection of dioxin-like compounds (DLCs) and aryl hydrocarbon receptor (AhR) agonists. This recombinant yeast reporter was based on a synthetic bacterial luciferase reporter gene cassette ( lux) that can produce the luciferase as well as the enzymes capable of self-synthesizing the requisite substrates for bioluminescent production from endogenous cellular metabolites. As a result, bioluminescent signal production is generated continuously and autonomously without cell lysis or exogenous reagent addition. By linking the expression of the autobioluminescent lux reporter cassette to AhR activation via the use of a dioxin-responsive promoter, the S. cerevisiae BLYAhS bioreporter emitted a bioluminescent signal in response to DLC exposure in a dose-responsive manner. The model dioxin, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), could be detected within 4 h with a half maximal effective concentration (EC) of ~ 8.1 nM and a lower detection limit of 500 pM. The autobioluminescent response of BLYAhS to other AhR agonists, including 2,3,7,8-tetrachlorodibenzofuran (TCDF), polychlorinated bisphenyl congener 126 (PCB-126) and 169 (PCB-169), 1,2,3,6,7,8-hexachlorodibenzo- p-dioxin (HxCDD), 1,2,3,4,6,7,8-heptachlorodibenzo- p-dioxin (HpCDD), benzo[a]pyrene (BaP), and β-naphthoflavone (bNF), were also characterized in this study. The non-destructive and reagent-free nature of the BLYAhS reporter assay facilitated near-continuous, automated signal acquisition without additional hands-on effort and cost, providing a simple and cost-effective method for rapid DLC detection. [ABSTRACT FROM AUTHOR]

Based on the sole information of structural genes of the 2-nitrobenzoate (2NBA) utilizing catabolic gene cluster (onbX1X2FCAR1EHJIGDBX3), 2NBA-sensing bioreporters were constructed by incorporating egfp into the onb gene cluster of Cupriavidus sp. strain ST- 14. Incorporation of reporter gene in proximal to the hypothesized promoter region in conjunction with the disruption of the gene encoding inducer-metabolizing enzyme was turned out to be advantageous in reporter gene expression at low inducer concentration. The bioreporter strain was capable of expressing EGFP from the very 1st hour of induction and could detect 2NBA at (sub) nanomolar level exhibiting a strict specificity toward 2NBA, displaying no response to EGFP expression from its meta- and paraisomers as well as from a number of structurally related compounds. The present study is a successful demonstration of the development of a 2NBA-sensing bioreporter with respect to ease of construction, inducer specificity, and sensitivity, without prior knowledge of the associated inducer-responsive promoter-regulator elements. The present approach can be used as a model for the development of bioreporters for other environmental pollutants. [ABSTRACT FROM AUTHOR]

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an 'omega-loop' motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin, altered emission colors, and altered substrate specificity for redshifted analog DL-infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox. [ABSTRACT FROM AUTHOR]

Aims Evaluate the use of polyethyleneimines (PEIs) as membrane permeabilizers to improve the responses and sensitivity of a bacterial bioreporter strain to viologens. Methods and Results The responses from E. coli str. EBS, i.e., E. coli BW25113 carrying plasmid pSDS, when exposed to five different viologens were characterized, as were the toxicities of seven different PEIS, including two linear and five branched species. Based on these results, benzyl viologen led to the greatest responses, and 0·8-kDa branched PEI (BPEI) was the least toxic of the PEIs tested and, therefore, both were selected for the subsequent tests. The bioluminescence and relative responses from E. coli str. EBS exposed to various concentrations of 0·8 kDa BPEI identified 400 mg l−1 as the optimal concentration. Using this concentration, tests were performed with all five of the viologens. Conclusions The responses from E. coli str. EBS to the viologens were improved, with the maximum relative bioluminescence values increasing between 5·6 and 16·5-fold. The minimum detectable levels for four of the viologens were likewise improved 2- to 4-fold. Significance and Impact of Study Improving bacterial membrane permeability in a controlled manner using BPEIs can improve biosensing of toxic compounds, as well as be used in biofuel and bioenergy applications where membrane permeability to a solute is important. [ABSTRACT FROM AUTHOR]

This study examined the applicability of a freeze-dried bioluminescent bioreporter, Pseudomonas putida mt-2 KG1206 (called KG1206), to the biomonitoring of groundwater samples. Samples were collected from the monitoring wells of gas station tanks or old pipeline leakage sites in Korea. In general, the freeze-dried strain in the presence of pure inducer chemicals showed low bioluminescence activity and a different activity order compared with that of the subcultured strain. The effects of KNO as a bioluminescence stimulant were observed on the pure inducers and groundwater samples. The stimulation rates varied according to the type of inducers and samples, ranging from 2.2 to 20.5 times (for pure inducers) and from 1.1 to 11 times (for groundwater samples) the total bioluminescence of the control. No considerable correlations were observed between the bioluminescence intensity of the freeze-dried strain and the inducer concentrations in the samples ( R < 0.1344). However, samples without a high methyl tertiary butyl ether (MTBE) level and those from the gas station leakage site showed reasonable correlations with the bioluminescence activity with R values of 0.3551 and 0.4131, respectively. These results highlight the potential of using freeze-dried bioluminescent bacteria as a rapid, simple, and portable tool for the preliminary biomonitoring of specific pollutants at contaminated sites. [ABSTRACT FROM AUTHOR]

Oil spill incidents occur frequently and threaten ecosystems and human health. Solid-phase microextraction allows direct alkane extraction from environmental matrices to improve the limit of detection but is unable to measure alkanes on site. A biological-phase microextraction and biosensing (BPME-BS) device was developed by immobilising an alkane chemotactic Acinetobacter bioreporter ADPWH_alk in agarose gel to achieve online alkane quantification with the aid of a photomultiplier. The BPME-BS device had a high enrichment factor (average 7.07) and a satisfactory limit of detection (0.075 mg/L) for alkanes. The quantification range was 0.1–100 mg/L, comparable to a gas chromatography flame ionisation detector and better than a bioreporter without immobilisation. ADPWH_alk cells in the BPME-BS device maintained good sensitivity under a wide range of environmental conditions, including pH (4.0–9.0), temperature (20–40 °C), and salinity (0.0–3.0%), and its response remained stable within 30 days at 4 °C. In a 7-day continual measurement, the BPME-BS device successfully visualised the dynamic concentration of alkanes, and a 7-day field test successfully captured an oil spill event, helping in source apportionment and on-scene law enforcement. Our work proved that the BPME-BS device is a powerful tool for online alkane measurement, showing substantial potential for fast detection and rapid response to oil spills on site and in situ. [Display omitted] • First alkane online detection device using immobilized bioreporters. • Detection limit of 0.075 mg/L and quantification range of 0.1–100 mg/L for alkanes. • Stable performance under pH 4.0–9.0, temperature 20–40 °C, salinity 0.0–0.3%. • Long shelf-life of 30 days at 4 °C. • Successful oil spill recognition in field test for water quality management. [ABSTRACT FROM AUTHOR]